Formulations comprising multiple dietary and endogenously made antioxidants and B-vitamins and use of same

ABSTRACT

The invention is directed to formulations comprised of multiple dietary and endogenously made antioxidants and B-vitamins and the use of these formulations in preventing and treating coronary artery disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of Provisional Application No.60/653,700 filed Feb. 17, 2005, the contents of which are specificallyincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to formulations comprised of multiple dietaryand endogenously made antioxidants and B-vitamins and the use of theseformulations in preventing and treating coronary artery disease.

2. Description of the Related Art

Coronary artery disease (CAD) remains the number one cause of death inthe U.S.A. About 1.5 million new cases are detected annually, andapproximately 1 million people die of this disease every year. Thecurrent estimate is that about 62 million Americans have one or moretypes of cardiovascular disease, and about 14 million per year sufferfrom heart attack or angina. The direct and indirect financial cost inthe U.S.A. of this disease is estimated to be about 329 billion dollarsannually.

Various organizations and health professionals have proposed primaryprevention strategies that involve changes in the diet and lifestyle.Although these recommendations appear rational, they have not had anysignificant impact in reducing the risk of heart disease. This may bedue to the fact that most people do not follow lifestyle changes anddiet modifications, and those who do follow them do not start untilatherosclerosis is well established. At present, statin with or withoutniacin is recommended to patients with high LDL-cholesterol and lowHDL-cholesterol, and aspirin for the primary prevention as well as forthe treatment of CAD. However, some patients at highercholesterol-lowering drug doses may exhibit muscle pain and livertoxicity, and a significant number of these patients develop varyingdegrees of aspirin resistance. Therefore, the current strategy for theprevention and treatment of CAD needs improvement.

Since increased oxidative stress and homocysteine level are consideredmajor risk factors in the etiology of CAD, it appears rational tosuggest that daily supplementation with multiple dietary andendogenously made antioxidants together with B-vitamins may enhance theefficacy of the current strategies for the prevention and treatment ofCAD. Although the U.S. Prevention Service Task Force recommends vitaminsupplements to reduce the risk of cancer and CAD, the role ofantioxidants alone or in combination with standard therapy in theprevention or treatment of CAD has become a controversial and confusingissue for the public as well as professionals. This is due to the factthat interventional trials with dietary antioxidants, mostly withvitamin E alone or in combination with cholesterol-lowering drugs, haveproduced inconsistent results, varying from a beneficial effect to noeffect to a harmful effect.

A few excellent reviews have summarized the published data on theseissues, but did not identify possible reasons for inconsistent resultsof interventional trials with antioxidants, and did not propose anyspecific recommendations for the prevention or as adjunct to standardtherapy for the treatment of CAD. Based on the current inconsistentresults of intervention studies, it is inappropriate to promote thenotion that antioxidants have no value in the prevention or treatment ofCAD or that they may have adverse effects in CAD patients taking statinsand/or niacin.

The following is a discussion of etiologic risk factors, andepidemiologic, laboratory and intervention studies with antioxidantsalone or in combination with statins and/or niacin. It identifies thepossible reasons for inconsistent results. The discussion also proposesa scientific rationale for why nutritional formulations may improve theefficacy of standard therapy in the prevention or treatment of CAD.

I. Etiologic Risk Factors for CAD

Several investigations have identified risk factors for the developmentof CAD. These include a family history, age, High LDL-cholesterol andlow HDL-cholesterol, high saturated fat intake, hypertension, physicalinactivity, cigarette smoking, obesity, diabetes mellitus, inflammatoryreactions and coronary calcium level. The combination of these riskfactors may increase the risk or the rate of progression of CAD morethen that produced by any one individual risk factor. One of themechanisms of most of these diverse risk factors, such as inflammatoryreactions, cigarette smoking and hyperglycemia, may involve theproduction of enhanced levels of free radicals. Increased levels of freeiron, copper or manganese, impaired mitochondrial function orantioxidant deficiency can further increase the production of freeradicals. Free radicals derived from oxygen and nitrogen, referred to asreactive oxygen species (ROS) and reactive nitrogen species (RNS), candamage lipids, proteins, RNA and DNA. Although certain levels of freeradicals are essential for driving the biological reactions necessaryfor human survival, excess levels of these free radicals can increasethe risk of most human chronic diseases, including CAD.

A. Oxidative Damage, C-reactive Proteins and Cholesterol

LDL-cholesterol is commonly referred to as a “bad cholesterol” becauseit is easily oxidizable by free radicals. Oxidized LDL-cholesterol maybe one of the early events that could initiate plaque formation byincreasing the formation of foam cells in the arterial wall; enhancingplatelet adhesion and aggregation, triggering thrombosis and impairingelasticity of the coronary arteries. Oxidized LDL-cholesterol can alsoincrease vascular smooth muscle cell proliferation by activating c-myc,and its binding partner MAX, and the carboxy-terminal domain-bindingfactors activator protein-2 (AP-2), and elongation 2 factor (E2F) inhuman coronary artery smooth muscle cells. The importance of c-myc inthe progression of atherosclerotic lesions is demonstrated by the factthat gene therapy, by decoy/oligodeoxynucleotide which inactivates E2F,delivered to human bypass vein grafts intraoperatively, caused fewergraft occlusions and critical stenosis after 12 months.

Oxy LDL-cholesterol is also engulfed by macrophages to form foam cells,and C-reactive protein increases the uptake of oxyLDL-cholesterol bymacrophages. Both foam cells and increased proliferation of vascularsmooth muscle cells contribute to the formation of plaque in thecoronary arteries. Thus lowering the level of LDL-cholesterol andpreventing the oxidation of this form of cholesterol should reduce theinitiation, as well as the progression, of CAD.

B. Endothelial Cells, Free Radicals and Homocysteine

Endothelial cells of the vascular wall are damaged by the free radicalsand the secretory products of inflammatory reactions (i.e., freeradicals, cytokines and prostaglandin E₂), as well as by homocysteine.Endothelial cell-derived nitric oxide (NO) is a potent vasodilator thatis formed during the metabolism of arginine, and it is responsible forregulating vascular tone. It is now recognized that endothelial celldysfunction may also be one of the early events in the development ofCAD.

Damage to endothelial cells may impair the nitric oxide synthase (NOS)pathway, which in turn may impair endothelium-dependent coronary arterydilation. Thus, deficiency in the production of NO may interfere withthe function of the vessel wall. The level of inducible NOS (iNOS) mRNAin vascular smooth muscle cells of a healthy arterial wall is low, butthe levels of iNOS mRNA and protein in macrophages and in the majorityof early lesions and in all advanced atherosclerotic lesions are high.This suggests that these sources may release an excessive amount of NOthat could increase the progression of coronary artery dysfunction.Thus, the role of NO in maintaining normal vascular function dependsupon maintaining proper levels of NO.

Both deficiency and excess production of NO can impair vasomotion andcan enhance endothelial dysfunction. In the case of deficiency,restoring the physiological level of NO can improve the endothelialdysfunction and vascular wall defect. Damaged endothelial cells reducethe release of nitric oxide (NO) which impairs the vasodilation ofarteries. Therefore, protecting the endothelial cells against freeradicals may be considered helpful in prevention or treatment strategy.Once the plaque is formed, it serves as a continuous stimulus forincreased inflammatory reactions that in turn causes a gradualprogressive increase in plaque size.

C. Development of Aspirin Resistance

Aspirin is commonly recommended for the primary prevention and treatmentof CAD, because it reduces inflammatory reactions and production ofprostaglandin E₂ (PGE₂) by inhibiting cyclooxygenase activity. PGE₂ isone of the major factors responsible for platelet aggregation. About5-12% of cardiac patients develop resistance to aspirin and about 24% ofpatients taking aspirin become semi-responders. The exact mechanisms ofaspirin resistance remain to be elucidated. It is possible that aspirindoses commonly used fail to suppress cyclooxygenase activity, requiringphysicians to increase aspirin doses. It is also possible that aspirinbecomes ineffective in reducing the activity of cyclooxygenase and/orthat PGE₂ is produced non-enzymatically. The latter would make patientstotally resistant to aspirin. It has been reported that the risk ofmajor cardiac events may increase by about 3-fold in aspirin resistantpatients. Therefore, resolving the issue of aspirin resistance hasbecome a new challenge for researchers in cardiology.

II. Role of Antioxidants in the Prevention and Treatment of CAD

In humans, different types of inorganic and organic free radicalsderived from oxygen and nitrogen are produced. Oxygen-derived freeradicals include O⁻.(anion), OH.(hydroxyl free radicals), O.(oxy freeradicals), RO.(peroxy free radical) and R.(organic free radical).Nitrogen-derived free radicals include NO.(nitric oxide) andNOOH.(peroxynitrite). Humans also produce certain antioxidants such asantioxidant enzymes, glutathione, coenzyme Q10, alpha lipoic acid andNADH (nicotinamide adenine dehydrogenase, reduced form). They alsoconsume antioxidants through their diet, such as vitamin A, vitamin C,vitamin E, beta-carotene and other carotenoids, phenolic compounds, andthe mineral selenium (which acts as a cofactor for glutathioneperoxidase).

These antioxidants, at least in part, have different affinities withvarious types of free radicals. With these complexities of relationshipbetween antioxidant and oxidant reactions, it is not rational to utilizeone or two antioxidants for the prevention or the treatment of anydiseases, including CAD. In addition, an individual antioxidant, when itbecomes oxidized, acts as a free radical. Therefore, the use of a singleantioxidant in, for example, heavy smokers (men or women), who have ahigh oxidative body environment, to reduce the risk of any chronicdiseases makes no scientific sense. Unfortunately, most human trials inCAD involve one or two dietary antioxidants without consideration ofinternal oxidative environment and this may be one of the major reasonsfor the inconsistent results.

Another reason could be that all intervention trials published thus fardo not give proper consideration to the form of dietary antioxidants(natural vs. synthetic vitamin E and β-carotene, with the natural formbeing more effective than the synthetic form), did not include bothvitamin and β-carotene (β-carotene having functions other than being aprecursor of vitamin A), did not include d-alpha tocopherol as well asd-α-tocopheryl succinate (α-tocopheryl succinate being the mosteffective form of vitamin E) and did not include endogenously madeantioxidants such as co-enzyme Q10, glutathione elevating agents(n-acetylcysteine) and alpha-lipoic acid.

A. Laboratory Studies

Most laboratory studies support the hypothesis that supplementation withantioxidants may prevent and reduce the rate of progression ofatherosclerosis. For example, Vitamin E supplementation has been knownto reduce the development of early aortic atherosclerotic lesions inchickens and rabbits. Further, stenosis progression was decreased inmale monkeys receiving vitamin E supplementation in comparison to thosereceiving an unsupplemented atherosclerosis-promoting diet. The abovestudies utilized vitamin E alone. A high oxidative internal environmentmay not exist in rodents; therefore, it is unlikely that adverse effectsof a single antioxidant can be observed in the animal model. This maynot be true in humans, especially in heavy smokers. In addition, unlikein humans, the lifestyle and diet are fairly constant in these animalmodels.

B. Proposed Mechanisms of Action of Antioxidants

Several mechanisms of action of antioxidants in reducing the risk of CADhave been proposed, although most studies have focused on the action ofvitamin E alone. For example, vitamin E reduces oxidation of membraneand LDL cholesterol, reduces c-myc activated pathways responsible forsmooth muscle cell proliferation, reduces aggregation of platelets innormal subjects, type I diabetes patients and heart transplantrecipients on cyclosporine, and inhibits the protein kinase C pathway.

Vitamin C prevents lipid oxidation and oxyLDL-cholesterol mediatedpathway. Vitamin C also potentiates nitric oxide activity in normalizingvascular function in patients with cardiac disease associated withhypercholesterolemia, hyperhomocysteinemia, hypertension and smoking.

The combination of vitamins C and E produces a synergistic inhibition onLDL-cholesterol oxidation.

Alpha-tocopheryl succinate inhibits the expression of c-myc in somemammalian cells, and α-tocopherol reduces the proliferation of vascularsmooth muscle cell proliferation in culture by inhibiting protein kinaseC activity that is independent of antioxidant properties.

Co-enzyme Q10 acts as a cofactor in the Krebs's cycle in mitochondria togenerate ATP.

Alpha-lipoic acid improves glucose utilization in peripheral tissue bystimulating glucose transport and uptake. Therefore, it can improve thefunction of damaged muscle or endothelial cells.

SH-compounds such as glutathione are one of the most importantintracellular antioxidants responsible for protecting cells against freeradical damage. Oxidative damage can reduce the level of SH-compoundswithin the cells. Therefore, glutathione-elevating agents such asN-acetylcysteine (NAC) and α-lipoic acid may be useful in restoringintracellular levels of glutathione in endothelial cells and cardiacmuscle cells, thereby improving the function of these cells andprotecting them from further oxidative damage.

Thus, mechanistic data exist that support the rationale for using amixture of dietary and endogenously made antioxidants in the prevention,and as an adjunct to standard therapy, in the treatment of CAD.

C. Epidemiological Studies

Epidemiological studies are a useful way to establish an associationbetween dietary nutrients including antioxidants and the risk of CAD.However, some inherent complexities of the varied human diet andlifestyle, techniques of data collection that rely on memory orshort-term dietary records and the multifactorial nature of CAD, make itdifficult for them to yield consistent results. Nevertheless, 6 out of 8epidemiologic studies that have been performed with vitamin E alone showan inverse relationship between vitamin E intake and the risk of CAD.

In particular, in a WHO/MONICA study there was a high inverseassociation between age-specific mortality from ischemic heart diseaseand lipid-standardized vitamin E levels. In a Polish study, plasmalevels of vitamin E were significantly lower in patients with stable andunstable angina compared to healthy control persons. In a UK study, aninverse association between plasma vitamin E levels and risk of anginawas reported. In a Harvard study of 39,910 male health professionals, a36% lower relative risk of CAD was demonstrated among those consuming 60IU vitamin E per day in comparison to those consuming less than 7.5 IUvitamin E per day. Men who took at least 100 IU vitamin E per day for atleast 2 years had a 37% lower risk of CAD than those who did not takevitamin E. Another Harvard study of 87,245 healthy nurses with afollow-up period of 8 years revealed that women in the top fifth ofvitamin E intake had a 34% lower relative risk of major CAD incomparison to those in the lowest fifth. The relative risk of CAD was48% lower in women taking vitamin E supplements of more than 100 mg perday for at least 2 years. Vitamin E obtained only from the diet providedno such protection. In another US study of 11,000 people of age 67 orover, with a follow up period of 6 years, it was found that vitamin Esupplementation was associated with a 47% reduction in mortality fromCAD. Further reduction was observed in people who were taking vitamin Esupplements together with vitamin C.

In contrast to the 6 investigations discussed above, 2 studies failed toobserve any association between serum selenium, vitamin A or vitamin Eand the risk of death from CAD. Because of numerous confounding factorsassociated with diet and lifestyle, epidemiologic studies have producedinconsistent results. It is surprising that most studies have showninverse relationship between vitamin E intake and the risk of CAD.

D. Intervention Studies in Humans

CAD is considered a multi-factorial disease that involves multiple riskfactors such as increased oxidation and inflammatory reactions, highlevels of LDL-cholesterol and low levels of HDL-cholesterol, as well ashigh levels of homocysteine and glucose. Each of these risk factors maycontribute variable degrees of risk to the initiation and progression ofCAD. Therefore, the purpose of any intervention trials should be toattenuate these risk factors for CAD. This would require amultifactorial approach involving, statin with or without niacin,multiple dietary and endogenously made antioxidants, B-vitamins andmineral selenium, and aspirin. Unfortunately, to date no such studieshave been performed.

The clinical studies that have been published have produced inconsistentresults. This is due to the fact that form, type, number, dose anddose-schedule of antioxidants, study end points, observation period andpatient population differ from one study to another. When antioxidantswere used in combination with cholesterol-lowering drugs, similarinconsistent results for the same reasons were also noted.

The published intervention trials can be divided into four groups, (a)those using a single dietary antioxidant in patients with no priorcardiac events; (b) those using a single dietary antioxidant in patientswith prior cardiac events; (c) those using two or more dietaryantioxidants with or without prior cardiac events; and (d) those usingone or more dietary antioxidants in combination withcholesterol-lowering drugs in patients with or without prior cardiacevents. Each of these is discussed below.

1.) Intervention Trials with One Dietary Antioxidant in Patients with NoPrior Cardiac Event

A summary of 5 intervention trials involving the use of one dietaryantioxidant in patients with no prior cardiac events is presented inTable 1 below. Data showed that dose and type of antioxidants,population type, study endpoint and follow-up period differ from onestudy to another. Nevertheless, three studies showed the beneficialeffects of dietary antioxidants, one study reported no beneficial effectand another investigation showed an adverse effect.

For example, vitamin E supplementation (544 IU) improvedendothelial-dependent flow-mediated dilation (FMD) of blood vessels insmokers with hypercholesterolemia. An elevation of plasma homocysteineby methionine impaired endothelial function in healthy male subjects.Pretreatment with vitamin C (2 g/day) prevents this effect and reducedarterial stiffness. In the Cambridge Heart Antioxidant Study (CHAOS)involving 2002 patients with angiographically proven coronaryatherosclerosis, it was found that patients receiving 400 or 800 I.U. ofvitamin E per day for a median follow-up period of 510 days (about 18months) had a 77% lower risk of non-fatal myocardial infarction than theplacebo group. However, a small (18%) but statistically significantincrease in cardiovascular death was observed in the vitamin E group.The investigators suggested that most of the deaths in the vitamin Egroup occurred in the early part of the follow-up period; and therefore,they were unlikely due to vitamin E.

In contrast to the above observation, vitamin E supplementation in theHOPE trial involving 9,541 patients with high risk for cardiac events(atherosclerotic disease, diabetes plus one cardiac risk factor such ashypertension, elevated total cholesterol, low HDL cholesterol, cigarettesmoking or documented microalbumineria), but without any evidence ofheart failure, did not reduce death, stroke or non-fatal myocardialinfarction in comparison to control. The exact reasons for thisdiscrepancy between CHAOS and HOPE trials are unknown. However, it waspointed out that the HOPE trial used vitamin E (400 IU) from naturalsources that had α-tocopherol and tocotrienol, and that the α-tocopherolportion was only about 50 IU. It was suggested that larger doses ofα-tocopherol may be required to reduce the inflammation andatherogenesis.

Another study showed that daily consumption of 800 I.U. of vitamin Eincreased the levels of oxidative stress markers in heavy smokers. Thiswas expected because vitamin E in the presence of an elevated level ofoxidative environment oxidized that is present in heavy smokers wasoxidized and acted as a free radical. Among non-smokers, vitamin E mayproduce transient benefits on certain criteria, but it would beineffective on a long-term basis. Therefore, both CHAOS and HOPE trialshave serious limitations, since they have utilized only vitamin E. Fromthe studies presented in this section, no conclusion regarding the valueof antioxidants for the prevention or treatment of CAD can be drawn.TABLE 1 Summary of intervention trials with vitamin E or vitamin C inhigh risk patients with no prior cardiac events Name of No. of Type ofCriteria of Follow-up Study Patients Antioxidant Study Period ResultsHOPE 9,541^(a)   Vitamin E Death, stroke 4.5 y no effect 400 IUNon-fatal MI CHAOS 2,002^(b)   Vitamin E (d-αT) Death, non-fatal 510 dreduced 400 or 800 IU MI — 42^(c) Vitamin E(α-TA) FMD 4 months improved544 IU — 10^(d) Vitamin E Oxidative stress 3 weeks increases 800 IU —80^(e) Vit. C-2g Plasma homocysteine, 6 hours reduced Methione-100 mg/kgarterial stiffnessHOPE (Heart Outcomes Prevention Evaluation);CHAOS (Cambridge Heart Antioxidant Study);MI (myocardial infarction);FMD (endothelial-dependent flow-mediated dilation)^(a)High risk for cardiac events (known atherosclerotic disease,diabetes plus one cardiac risk factor such as hypertension, elevatedtotal cholesterol, low HDL cholesterol, cigarette smoking or documentedmicroalbumineria), and not have heart failure.^(b)Proven atherosclerotic disease^(c)Hypercholesterolemia, smokers and smokers with hypercolesterolemia^(d)Heavy smokers^(e)Healthy male subjects receiving methionine to increase homocysteinelevels2.) Intervention Trials Utilizing Only Vitamin E in Patients with PriorCardiac Events

A summary of 4 intervention trials using only vitamin E in high-riskpatients with prior cardiac events is presented in Table 2 below.Results showed that doses of vitamin E, patient population, studyendpoint and follow-up periods differ from one investigation to another.Nevertheless, three of four studies showed beneficial effects of vitaminE and one study revealed that the use of a low dose of vitamin E wasineffective.

For example, in a trial involving 100 patients having coronaryangioplasty, those receiving 1200 I.U. of vitamin E daily for a 4-monthperiod showed restenosis in 34.6% of patients, whereas those receiving aplacebo showed stenosis in 50% of patients. In another study involving75 patients, those receiving α-tocopherol (1200 IU/day) for a period of5 months had lowered C-reactive proteins and monocyte interleukin-6 indiabetes type 2 patients with or without macrovascular complications incomparison to BMI-matched healthy controls. In still another study,supplementation with α-tocopherol (800 IU) decreased LDL-C oxidation inpatients with chronic renal failure, but the benefit was greater inpatients with peritoneal dialysis than that in hemodialysis.

In contrast to the above three studies, results of the ATBC trialinvolving 1862 men for a period of 6 years showed no significantdifference in the number of major coronary events or cardiovasculardeath between the vitamin E group (50 mg/day) and placebo group. Thisstudy has utilized a much lower dose of vitamin E than the previous fourstudies. This could explain the above inconsistency in results. Again,using only one dietary antioxidant in these trials may not besignificant for cardiac prevention or treatment. TABLE 2 Summary ofintervention trials with vitamin E alone in high risk patients withprior cardiac events Name of No. of Type of Criteria Follow-up StudyPatients Antioxidant of Study Period Results — 100^(a)  Vitamin EStenosis 4 months reduced 1200 IU — 75^(b) Vitamin E C- reactive 5months reduced 1200 IU protein — 33^(c) Vit. E (d-αT) LDL 12 weeksreduced 800 IU oxidation ATBC 1862^(d)  Vitamin E Death, CAD 6 y noeffect 50 mg eventsAll vitamins were given once a day until specified otherwise^(a)Angioplasty^(b)Type II diabetes with or without macrovascular complications andBMI-matched healthy control^(c)Patients undergoing peritoneal dialysis (N = 17) or hemodialysis (N= 16)^(d)Male heavy smokers who have a previous history of myocardialinfarction3.) Intervention Trials Utilizing Two or More Antioxidants in Patientswith or without Prior Cardiac Events

A summary of 9 interventional trials with two or more antioxidants inhigh risk patients with or without prior cardiac events is presented inTable 3 below. Results showed that dose, type and number ofantioxidants, patient population, study endpoint and follow-up periodsdiffer from one study to another. Nevertheless, six studies showedbeneficial effects, one study revealed no beneficial effect while tworevealed harmful effects.

In a double-blind randomized prospective study, supplementation with 500mg vitamin C and 400 IU vitamin E twice daily retarded early progressionof transplant associated coronary arteriosclerosis. Supplementation withvitamin E and slow release vitamin C also reduced atherosclerosis.Another study has reported that supplementation with tomato juice (500ml/day), vitamin C (500 mg/day) and vitamin E (800 IU/day) decreasedoxidation of LDL cholesterol and plasma levels of C-reactive proteins intype 2 diabetes patients. It has been suggested that vitamin C may slowatherogenesis by improving endothelial-dependent vasodilation inpatients with hyperglycemia and abnormal lipids, perhaps by preventingoxidation by nitric oxide. Pretreatment with 800 I.U. of vitamin E and1000 mg of vitamin C prevented the above action of a high fat diet onendothelium-dependent vasodilation of the brachial artery. A clinicaltrial involving 317 patients who received a placebo or multipleantioxidants (700 IU vitamin E, 500 mg vitamin C and 30,000 IUbeta-carotene ) or probucol 4 weeks before and 6 months after undergoingangioplasty procedures, showed no significant difference in the rates ofrestenosis between the vitamin group and placebo group; however,probucol, a powerful antioxidant, was effective in reducing restenosis.Another study has shown that supplementation with multiple vitaminsreduced the homocysteine level and LDL-cholesterol oxidation. AmongCARET participants, supplementation with 30 mg β-carotene and 25,000I.U. retinyl palmitate for a 5-year period did not change lipid levels.This may be due to the fact that only vitamin A and its precursor wereused in this study and this may not be sufficient to alter lipid levels.In the ATBC trial involving 1862 male heavy smokers, supplementationwith 50 mg vitamin E and 20 mg β-carotene for a period of 5.3 yearsincreased major cardiac risks. This may due to the fact that doses ofthese antioxidants were low and male heavy smokers had high levels ofoxidative environment. This could have resulted in rapid oxidation ofthese antioxidants, thereby increasing the CAD risk factors.Supplementation with vitamin C (500 mg/day) and vitamin E (800 IU/day)increased the risk of atherosclerosis among postmenapausal women with ahistory of varying levels of stenosis. Again, no conclusion regardingthe value of antioxidants in prevention or treatment of CAD can be made,because endogenously made antioxidants were missing from these trials.Even the doses and number of dietary antioxidants varied from one studyto another. TABLE 3 Summary of intervention trials with two or moreantioxidants in high risk patients with or without prior cardiac eventsName of No. of Type of Criteria of Follow-up Study Patients AntioxidantStudy Period Results — 19^(c) Vit. E-400 IU Coronary 1 y reduced Vit.C-500 mg Atherosclerosis — 520^(f)   Vit. E, slow Atherosclerosis 6 yreduced release vit. C — tomato juice 15 ml/d, vit C OxyLDL-C, decreased500 mg, vit E 800 IU C-reactive protein — 20^(e) Vitamin E 800 IUVitamin C-1 g FMD 6 h increased — 317^(d)  Probucol- 500 mg VitaminE-700 IU Restenosis 5-7 mo probucol reduced more Vitamin C-500 mgβ-carotene-30,000 I than combination 182^(f)   Multivitaminshomocysteine, 6 mo Reduced LDL-C oxidation CARET 52^(a) β-Carotene-30 mgRetinyl Lipid levels 5 y no effect palmitate-25000 IU ATBC 1862^(b)  VitE (α-T)-50 mg Major cardiac 5.3 y increased β-carotene-20 mg Wave423^(g)  Vit E 800 IU; vitamin C 1 g Stenosis 2.8 y increasedCARET (Carotene Retinol Efficacy Trial);ATBC (Alpha-Tocopherol Beta-Carotene Cancer Prevention Study);WAVE (Women's Angiographic Vitamin and Estrogen);FMD (endothelial-dependent flow-mediated dilation).All vitamins were given once a day until specified otherwise.^(a and b)= male heavy smokers;^(c)= cardiac transplant;^(d)= angioplasty;^(e)= normal individuals consuming high-fat meal;^(f)= hypercholestrolemic;^(g)= post-menopausal women with 15-75% stenosis;^(h)= type II diabetes patients.4.) Intervention Trials Utilizing One or More Antioxidants inCombination with Cholesterol-Lowering Drugs in Patients with or withoutPrior Cardiac Events

A summary of 6 interventional trials with one or more antioxidants incombination with cholesterol-lowering drugs in high risk patients withor without prior cardiac events is described in Table 4 below. Thenumber, type, dose and dose schedules of antioxidants, patientpopulation, observation period and criteria of study were different.Three studies revealed beneficial effects, one showed no beneficialeffects, whereas two investigations by the same group showed deleteriouseffects when combined with simvastatin-niacin.

It has been reported that vitamin E supplementation (300 IU/day)together with simvastatin for an 8 week period improvedendothelial-dependent flow-mediated dilation (FMD) as well asendothelial-dependent nitroglycerine-mediated dilation (NMD) in thebrachial artery of patients with hypercholesterolemia more that thatproduced by simvastatin alone. A short-term beneficial effect may beobtained by high doses of a single antioxidant. Indeed, it has beenreported that coenzyme Q₁₀ has been shown to be useful in improving thefunction of damaged cardiac muscle associated with congestive heartfailure and idiopathic dilated cardiomyopathy. In these instances,coenzyme Q₁₀ probably improves the mitochondrial function of damagedcardiac muscles and protects them from further injury.

One study using 156 men with previous coronary bypass surgery who werereceiving a cholesterol-lowering drug combination (colestipol-niacin)alone or in combination with vitamin E 100 I.U. per day showed that thevitamin E treated group revealed less progression of the narrowing oftheir coronary arteries in comparison to cholesterol-lowering drugsalone during a 4-year trial period. In The Heart Protection Studyinvolving 20,500 patients with high risk, supplementation withantioxidants vitamins (vitamin E 650 mg, vitamin C 250 mg and β-carotene20 mg) together with simvastatin for a period of 5.5-years did notinterfere with the beneficial effect of this statin. Doses ofantioxidants were small in this study, and endogenously madeantioxidants were not included in the antioxidant regimen. Oppositeresults were obtained by recent clinical studies in which vitamin C,1000 mg, vitamin E as d-α-tocopherol, 800 IU, natural beta-carotene, 25mg and selenium, 100 μg per day were given together withmevastatin-niacin in a subset of patients with low level ofHDL-cholesterol. Results revealed that niacin-induced elevation of HDLcholesterol was blocked by antioxidant supplements. This has not beenreproduced by another clinical study; and therefore, the value of thisinvestigation remains uncertain. In addition, dietary antioxidantsvitamin A and β-carotene, and endogenously made antioxidants,n-acetylcysteine (a glutathione-elevating agent), α-lipoic acid, andco-enzyme Q10 as well as B-vitamins were missing from their formulation.

The same group of investigators, using the same formulation, reportedthat a mixture of dietary antioxidants reduced the degree of proximalartery stenosis in comparison to placebo controls. However, incombination with simvastatin-niacin it was less effective than thesimvastatin-niacin group. Because of the small sample size (40 patientsper group) and unusually large variation in the results (200-700%variation around the mean value), no conclusion regarding the value ofantioxidants in the prevention or treatment of CAD can be drawn. Thesame group using the same mixture of dietary antioxidants reported thattreatment of patients with simvastatin plus niacin alone or with thedietary antioxidants significantly reduced total cholesterol,LDL-cholesterol and triglycerides, and increase HDL-cholesterol levelsin comparison to those who were not treated. There was no significantdifference between those receiving simvastatin plus niacin or thosereceiving simvastatin, niacin and antioxidants.

Analysis of plasma levels of noncholesterol sterols suggested thatlathosterol was directly and β-sitosterol was inversely related tostenosis. It was interesting to note that treatment with simvastatinplus niacin or simvastatin, niacin and antioxidants, decreases the levelof lathosterol and increases the level of β-sitolsterol to similardegrees. These data suggest that newly identified predictors of stenosiswere not significantly affected by the addition of antioxidants to thegroup receiving simvastatin plus niacin. Nevertheless, the investigatorsconcluded that antioxidants prevent the efficacy of simvastatin plusniacin treatment.

Most intervention studies have recommended taking supplements once aday. Based on the biological-half life of antioxidants that varies from6-10 h, the recommendation of taking antioxidants once a day may notproduce an optimal effect on the prevention or as an adjunct to standardtherapy in the treatment of CAD. The two clinical studies have attractedthe attention of cardiologists and primary care physicians, and have ledthem to believe that antioxidants have either no value or may adverselyaffect the strategy for cardiac prevention and treatment. However, theanalysis of their data suggests that such conclusion may be premature.Based on conflicting results of intervention trials with one or moredietary antioxidants and lack of data on appropriately designed clinicaltrials with multiple dietary and endogenously made antioxidants, it isnot appropriate to propagate an idea that antioxidants may harm patientswith cardiac risks, and that they have no role in prevention ortreatment of CAD. TABLE 4 Summary of intervention trials with one ormore antioxidants in combination with cholesterol-lowering drugs in highrisk patients with or without prior cardiac events Name of No. of Typeof Antioxidant + Criteria of Follow-up Study PatientsCholesterol-lowering drugs Study period Results —  7^(a) Simvastatin +FMD NMD 8 weeks improved vit. E-300 IU — 126^(b) Standard therapy +Cardiac muscle 6 y improved Co-enzyme Q10 function 33.3 mg (thrice/day)CLAS 156^(c) Colestipol + niacin Progressive 2 y reduced Vit. E- 100 IUor more Atherosclerosis HPS 20,500^(d)   Simvastatin + Vit. E-650 mgCardiac events 5.5 y no better than than drug Vit. C-250 mg −carotene-20 mg alone HATS 160^(e) Simvastatin + niacin + Vit. Stenosis 3y reduced drug effectiveness E-800 IU, Vit. C-1 g − but was moreeffective than carotene- 25 mg, control selenium-100 mcg (N = 46) HATS153^(e) Same HDL 1 y reduce drug effectivenessCLAS (Cholesterol-Lowering Atherosclerosis Study);HPS (Heart Protection Study);HATS (HDL-atherosclerosis Treatment Study);FMD (endothelium-dependent flow-mediated dilation);NMD (endothelium-independent nitroglycerine-mediated dilation);HDL- (high density lipoprotein cholesterol);N (sample size for the group).All vitamins were given once a day until specified otherwise;^(a)= hypercholesterolemia;^(b)= idiopathic dilated cardiomyopathy;^(c)= coronary bypass surgery;^(d)= women, elderly diabetics, people with low baseline cholesterolpre-treatment and those with prior occlusive non-coronary vasculardisease;^(e)= coronary disease with low HDL- cholesterol.

III. Scientific Rationale For Using Multiple Dietary and EndogenouslyMade Antioxidants, and B-vitamins and Minerals in the Prevention Or asan Adjunct To Standard Theraphy in the Treatment of CAD

Since increased production of free radicals may be involved in theinitiation and progression of CAD, the use of antioxidants appears to bea rational choice for the prevention and treatment of this disease. Theuse of single antioxidants for the prevention or treatment of CAD is notrecommended because antioxidants act as free radicals when oxidized.Instead, the present invention, as described below, involves the use offormulations incorporating multiple dietary and endogenously madeantioxidants because many different types of free radicals are producedand each antioxidant has a different mechanism of action and has adifferent affinity for each of these free radicals, depending upon thecellular environment.

For example, β-carotene (BC) is more effective in quenching oxygenradicals than most other antioxidants. BC can perform certain biologicalfunctions that cannot be carried out by its metabolite, vitamin A, andvice versa. In particular, it has been reported that BC treatmentenhances the expression of the connexin gene, which codes for a gapjunction protein in mammalian cells in culture, whereas vitamin Atreatment does not produce such an effect. Vitamin A can induce celldifferentiation in certain normal and cancer cells, whereas BC does not.The gradient of oxygen pressure varies within the cells. Vitamin E ismore effective as a quencher of free radicals in reduced oxygenpressure, whereas BC and vitamin A are more effective in higheratmospheric pressure. Vitamin C is necessary to protect cellularcomponents in aqueous environments, whereas carotenoids, vitamins A andE protect cellular components in non-aqueous environments. Vitamin Calso plays an important role in maintaining cellular levels of vitamin Eby recycling the vitamin E radical (oxidized) to the reduced(antioxidant) form.

Also, the oxidative DNA damage produced by oxidized vitamin C could beameliorated by vitamin E. The form and type of vitamin E used are alsoimportant in any clinical trial. It is known that various organs of ratsselectively absorb the natural form of vitamin E. It has beenestablished that alpha tocopheryl-succinate (α-TS) is the most effectiveform of the vitamin. The inventors have reported that oral ingestion ofα-TS (800 I.U./day) in humans increased plasma levels of not onlyα-tocopherol, but also of α-TS, suggesting that α-TS can be absorbedfrom the intestinal tract before hydrolysis to α-tocopherol. Selenium isa co-factor of glutathione peroxidase, and Se-glutathione peroxidasealso acts as an antioxidant. Therefore, selenium supplementationtogether with other antioxidants is also essential.

Glutathione, an endogenously produced compound, represents a potentintracellular protective agent against damage produced by free radicals.It catabolizes H₂O₂ and anions. However, oxidized glutathione may makethe cell more sensitive to further oxidative injuries. Therefore,increasing the intracellular levels of glutathione in vascularendothelial cells, vascular smooth muscle cells and cardiac muscles, maybe very useful in the management of CAD. Oral supplementation withglutathione failed to significantly increase plasma levels ofglutathione in human subjects, suggesting that this tripeptide iscompletely hydrolyzed in the G.I. tract. N-acetylcysteine and α-lipoicacid increase the intracellular levels of glutathione, and therefore,they can be used in combination with dietary antioxidants.

Furthermore, damaged vascular endothelial cells and cardiac muscle cellsmay not produce sufficient amounts of ATP due to a reduction in thesynthesis of coenzyme Q10. Therefore, supplementation with coenzyme Q10may be necessary for prevention, and as an adjunct to standard therapyfor the treatment of CAD. In addition to being a weak antioxidant,coenzyme Q10 acts as a co-factor for generating ATP in the mitochondria.It also scavenges peroxy radicals faster than α-tocopherol, and likevitamin C, can regenerate vitamin E in a redox cycle. B-6, B12 and folicacid are essential for reducing the level of homocysteine.

IV. Antioxidants and Aspirin Resistance

It has been reported that antioxidants in combination with aspirin ismore effective in inhibiting cyclooxygenase activity than either agentadministered by itself. Alpha tocopherol succinate has been shown toreduce the action of PGE₂ on adenylate cyclase. Thus, antioxidants canprolong the efficacy of aspirin among semi-responders and may preventplatelet aggregation among those who have developed total resistance.

DESCRIPTION OF CERTAIN EXAMPLARY EMBODIMENTS OF THE INVENTION

The discussion below describes certain exemplary embodiments offormulations produced in accordance with the present invention. Inaddition, it further discusses the scientific rationale behind theinventors' inclusion of multiple dietary and endogenously madeantioxidants, and B-vitamins and minerals in the formulations of theinvention, which formulations have been determined by the inventors tobe useful in preventing CAD, and/or as an adjunct to standard therapy inthe treatment of CAD:

Statin with or without niacin, and aspirin are considered standardtherapy for the prevention and treatment of CAD. The invention describedand claimed herein comprises nutritional formulations specific to mediumrisk patients (patients taking medications but had no cardiac events)and high risk patients (patients taking medications with one or morecardiac events). The formulations of the invention contain multipledietary and endogenously made antioxidants, B-vitamins including B-6,B-12 and folic acid and minerals including selenium (selenomethionine)that would enhance the efficacy of standard therapy for the preventionand treatment of CAD.

In one embodiment, the invention provides a formulation and a method forat least one of substantially preventing and treating coronary arterydisease in an individual in need of the same, which method comprisesadministering to such individual a therapeutically effective amount of aformulation comprising the following components in about the followingranges. As used herein, the term “about” shall be construed to mean±10%. Total Dose/day Vitamin A (palmitate) 3,000-5,000 I.U.Beta-carotene (from natural D. salina) 10-20 mg Vitamin D-3(cholecalciferol) 400-600 I.U. Natural source Vitamin E (d-alphatocopherol) 50-400 I.U. (d-alpha tocopheryl acid succinate) 100 I.U.Buffered Vitamin C (calcium ascorbate) 100-4,000 mg Thiamine mononitrate2-10 mg Riboflavin 2-20 mg Niacinamide ascorbate 15-200 mg d-calciumpantothenate 5-30 mg Pyridoxine hydrochloride 2-10 mg Cyanocobalamin5-20 mcg Folic Acid (Folacin) 400-1,200 μg D-Biotin 100-500 mcg Selenium(l-seleno-methionine) 50-200 mcg Chromium picolinate 50-200 mcg ZincGlycinate 10-30 mg Calcium citrate 100-500 mg Magnesium citrate 100-250mg Vitamin C 100-4,000 mg d-α-tocopheryl succinate 50-400 IUN-acetylcysteine 100-500 mg Alpha-lipoic acid 15-100 mg Coenzyme Q1010-250 mg Omega-3 fatty acid 1,000-2,000 mg

Example embodiments of recommended ingredients and doses of the proposednutritional formulations is presented below in Table 5. Because of thevariation in the biological half-life of antioxidants (6-10 h), it isrecommend (but not required) to ingest the formulation twice per day,i.e., half in the morning and half in the evening, before a meal. Theproposed doses are safe. TABLE 5 Recommended doses of dietary andendogenously made antioxidants, B-vitamins and selenium for theprevention and treatment of CAD Risk level Recommendation per day Mediumrisk SEVAK ®¹, a multiple dietary antioxidant with B- Vitamins andselenomethionine, but, with no Fe, Cu or Mn. In addition to SEVAK ®¹,the formulation contains vitamin C, about 1000 mg; d-α-tocopherylsuccinate, about 200 IU; N-acetylcysteine, about 250 mg; alpha- lipoicacid, about 30 mg; coenzyme Q10, about 30 mg; and omega -3 fatty acid,about 1000 mg. High risk SEVAK ®¹, a multiple dietary antioxidant withB- Vitamins, selenomethionine, but no Fe, Cu or Mn. In addition toSEVAK ®¹, the formulation contains vitamin C, about 2000 mg;d-α-tocopheryl succinate, about 600 IU; N-acetylcysteine, about 500 mg;alpha-lipoic acid, about 60 mg; coenzyme Q10, about 90 mg; and omega-3fatty acid, about 2000 mg. Vitamin A (palmitate) 5,000 I.U.Beta-carotene (from natural D. salina) 15 mg Vitamin D-3(cholecalciferol) 400 I.U. Natural source Vitamin E (d-alpha tocopherol)100 I.U. (d-alpha tocopheryl acid succinate) 100 I.U. Buffered Vitamin C(calcium ascorbate) 500 mg Thiamine mononitrate 4 mg Riboflavin 5 mgNiacinamide ascorbate 30 mg d-calcium pantothenate 10 mg Pyridoxinehydrochloride 5 mg Cyanocobalamin 10 mcg Folic Acid (Folacin) 800 mcgD-Biotin 200 mcg Selenium (l-seleno-methionine) 100 mcg Chromiumpicolinate 50 mcg Zinc Glycinate 15 mg Calcium citrate 250 mg Magnesiumcitrate 125 mgMedium risk: Patients taking medications without any cardiac events;High risk: patients taking medications with one or more cardiac events.¹The formulation of SEVAK ®, which is commercially available fromPremier Micronutrient Corporation, the owner of the present application,comprises the following components in about the following amounts:

The inventors additionally recommend a diet low in fat, and high infiber and antioxidants, regular exercise, reduced stress and coffeeconsumption, and no tobacco smoking.

Doses of antioxidants higher than those proposed in these formulationshave been used to treat human diseases without any toxic effects.Nevertheless, it is important to describe briefly the known doses ofantioxidants that could produce some toxicity. Daily consumption ofvitamin A at doses higher than twice the Recommended Daily Allowance(RDA) could produce birth defects in pregnant women, and doses 25,000IU/day or more can produce liver and skin toxicity after prolongedconsumption in adults. Vitamin C at doses of 10 g/d or more, whenconsumed in a single dose in the form of ascorbic acid, can cause upsetstomach and diarrhea in some cases. Persons with kidney disease andhemochromotosis may have adverse effects to high doses of vitamin C.Vitamin E doses up to 2,000 IU/d have been used for short-term clinicalstudies without adverse effects; however, intravenous administration of2,000 IU/d can cause clotting disorders which are reversible by vitaminK. Beta-carotene doses of 100 mg/d or more can cause yellowing of skinand pigment deposits in the eye which are reversible upondiscontinuation. N-acetylcysteine at doses of 1000 mg/day or more cancause trace metal deficiencies. Coenzyme Q10 doses up to 300 mg/d ormore have no known toxicity in humans.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. Thepresent invention, therefore, is not limited by the specific disclosureherein, but only by the claims.

1. A formulation for at least one of substantially preventing andtreating coronary artery disease in an individual in need of the same,which formulation comprises: Total Dose/Day Vitamin A (palmitate)3,000-5,000 I.U. Beta-carotene (from natural D. salina) 10-20 mg VitaminD-3 (cholecalciferol) 400-600 I.U. Natural source Vitamin E (d-alphatocopherol) 50-400 I.U. (d-alpha tocopheryl acid succinate) 100 I.U.Buffered Vitamin C (calcium ascorbate) 100-4,000 mg Thiamine mononitrate2-10 mg Riboflavin 2-20 mg Niacinamide ascorbate 15-200 mg d-calciumpantothenate 5-30 mg Pyridoxine hydrochloride 2-10 mg Cyanocobalamin5-20 mcg Folic Acid (Folacin) 400-1,200 mcg D-Biotin 100-500 mcgSelenium (l-seleno-methionine) 50-200 mcg Chromium picolinate 50-200 mcgZinc Glycinate 10-30 mg Calcium citrate 100-500 mg Magnesium citrate100-250 mg Vitamin C 100-4,000 mg d-α-tocopheryl succinate 50-400 IUN-acetylcysteine 100-500 mg Alpha-lipoic acid 15-100 mg Coenzyme Q1010-250 mg Omega-3 fatty acid 1,000-2,000 mg


2. A method for at least one of substantially preventing and treatingcoronary artery disease in an individual in need of the same, whichmethod comprises administering to such individual a therapeuticallyeffective amount of a formulation comprising: Total Dose/Day Vitamin A(palmitate) 3,000-5,000 I.U. Beta-carotene (from natural D. salina)10-20 mg Vitamin D-3 (cholecalciferol) 400-600 I.U. Natural sourceVitamin E (d-alpha tocopherol) 50-400 I.U. (d-alpha tocopheryl acidsuccinate) 100 I.U. Buffered Vitamin C (calcium ascorbate) 100-4,000 mgThiamine mononitrate 2-10 mg Riboflavin 2-20 mg Niacinamide ascorbate15-200 mg d-calcium pantothenate 5-30 mg Pyridoxine hydrochloride 2-10mg Cyanocobalamin 5-20 mcg Folic Acid (Folacin) 400-1,200 mcg D-Biotin100-500 mcg Selenium (l-seleno-methionine) 50-200 mcg Chromiumpicolinate 50-200 mcg Zinc Glycinate 10-30 mg Calcium citrate 100-500 mgMagnesium citrate 100-250 mg Vitamin C 100-4,000 mg d-α-tocopherylsuccinate 50-400 IU N-acetylcysteine 100-500 mg Alpha-lipoic acid 15-100mg Coenzyme Q10 10-250 mg Omega-3 fatty acid 1,000-2,000 mg


3. A method for at least one of substantially preventing and treatingcoronary artery disease in a medium risk individual in need of the same,which method comprises administering to such medium risk individual atherapeutically effective amount of a formulation comprising: TotalDose/Day Vitamin A (palmitate) about 5,000 I.U. Beta-carotene (fromnatural D. salina) about 15 mg Vitamin D-3 (cholecalciferol) about 400I.U. Natural source Vitamin E (d-alpha tocopherol) about 100 I.U.(d-alpha tocopheryl acid succinate) about 100 I.U. Buffered Vitamin C(calcium ascorbate) about 500 mg Thiamine mononitrate about 4 mgRiboflavin about 5 mg Niacinamide ascorbate about 30 mg d-calciumpantothenate about 10 mg Pyridoxine hydrochloride about 5 mgCyanocobalamin about 10 mcg Folic Acid (Folacin) about 800 mcg D-Biotinabout 200 mcg Selenium (l-seleno-methionine) abut 100 mcg Chromiumpicolinate about 50 mcg Zinc Glycinate about 15 mg Calcium citrate about250 mg Magnesium citrate about 125 mg Vitamin C about 1,000 mgd-alpha-tocopheryl-succinate about 200 I.U. N-acetylcysteine about 250mg Alpha-lipoic acid about 30 mg Coenzyme Q₁₀ about 30 mg Omega-3 fattyacid about 1,000 mg


4. A method for at least one of substantially preventing and treatingcoronary artery disease in a high risk individual in need of the same,which method comprises administering to such high risk individual atherapeutically effective amount of a formulation comprising: TotalDose/Day Vitamin A (palmitate) about 5,000 I.U. Beta-carotene (fromnatural D. salina) about 15 mg Vitamin D-3 (cholecalciferol) about 400I.U. Natural source Vitamin E (d-alpha tocopherol) about 100 I.U.(d-alpha tocopheryl acid succinate) about 100 I.U. Buffered Vitamin C(calcium ascorbate) about 500 mg Thiamine mononitrate about 4 mgRiboflavin about 5 mg Niacinamide ascorbate about 30 mg d-calciumpantothenate about 10 mg Pyridoxine hydrochloride about 5 mgCyanocobalamin about 10 mcg Folic Acid (Folacin) about 800 mcg D-Biotinabout 200 mcg Selenium (l-seleno-methionine) about 100 mcg Chromiumpicolinate about 50 mcg Zinc Glycinate about 15 mg Calcium citrate about250 mg Magnesium citrate about 125 mg Vitamin C about 2,000 mgd-alpha-tocopheryl-succinate about 600 I.U. N-acetylcysteine about 500mg Alpha-lipoic acid about 60 mg Coenzyme Q₁₀ about 90 mg Omega-3 fattyacid about 2,000 mg